The present invention relates to a method for producing L-phenylalanine, in particular, to a bacterium belonging to the genus Methylophilus and having an ability to produce L-phenylalanine and a method for producing L-phenylalanine using a bacterium belonging to the genus Methylophilus.
As a method for producing L-phenylalanine by using a microorganism by fermentation, those using recombinant bacteria belonging to the genus Escherichia (Japanese Patent Application Laid-Open Nos. 56-1890, 57-170184, 58-103398, 61-92565 and 1-104160, and International Publication No. WO 87/00202) are known. As a method for producing L-phenylalanine or L-tyrosine, one using a mutant belonging to the genus Corynebacterium (Japanese Patent Application Laid-Open No. 61-128897), and those using recombinant bacteria belonging to the genus Corynebacterium (Japanese Patent Application Laid-Open Nos. 60-34197, 60-24192, 61-260892, and 61-124375) are known.
However, production of L-phenylalanine by using a bacterium belonging to the genus Methylophilus is not known.
An object of the present invention is to provide a bacterium having an ability to produce L-phenylalanine and a method for producing L-phenylalanine by using the bacterium.
The present inventors have found that a bacterium belonging to the genus Methylophilus which is resistant to a phenylalanine analog, has a high ability to produce L-phenylalanine. Based on the finding, the present invention has been accomplished.
The present invention provides a bacterium belonging to the genus Methylophilus, which has an ability to produce L-phenylalanine and is resistant to a phenylalanine analog (hereinafter, also referred to as xe2x80x9cbacterium of the present inventionxe2x80x9d).
The bacterium of the present invention is preferably resistant to the phenylalanine analog and L-phenylalanine.
The bacterium of the present invention is preferably resistant to DL-p-fluorophenylalanine.
The bacterium of the present invention is preferably obtained by selecting a strain which is resistant to the phenylalanine analog and L-phenylalanine from bacteria belonging to the genus Methylophilus, wherein said selection is performed at least once for each of the phenylalanine analog and L-phenylalanine in any order.
The bacterium of the present invention is more preferably resistant to DL-p-fluorophenylalanine and m-fluorophenylalanine, as well as to L-phenylalanine.
The bacterium of the present invention is more preferably obtained by selecting a strain which is resistant to DL-p-fluorophenylalanine, m-fluorophenylalanine and L-phenylalanine from bacteria belonging to the genus Methylophilus, wherein said selection is performed at least once for each of DL-p-fluorophenylalanine, m-fluorophenylalanine and L-phenylalanine in any order.
The bacterium of the present invention is preferably resistant to DL-p-fluorophenylalanine, m-fluorophenylalanine and cinnamic acid, as well as L-phenylalanine.
The bacterium of the present invention is more preferably obtained by selecting a strain which is resistant to DL-p-fluorophenylalanine, m-fluorophenylalanine, cinnamic acid and L-phenylalanine from bacteria belonging to the genus Methylophilus, wherein said selection is performed at least once for each of DL-p-fluorophenylalanine, m-fluorophenylalanine, cinnamic acid and L-phenylalanine in any order.
The bacterium of the present invention is preferably Methylophilus methylotrophus. 
The present invention also provides a method for producing L-phenylalanine, comprising the steps of:
culturing the bacterium of the present invention in a culture medium to produce and accumulate L-phenylalanine in a culture, and
recovering L-phenylalanine from the culture (hereinafter, also referred to as xe2x80x9cmethod of the present invention).
 less than 1 greater than  The Bacterium of the Present Invention
The bacterium of the present invention is a bacterium belonging to the genus Methylophilus, which has an ability to produce L-phenylalanine and is resistant to a phenylalanine analog, and preferably to the phenylalanine analog and L-phenylalanine.
The bacterium belonging to the genus Methylophilus includes Methylophilus methylotrophus. 
The ability to produce L-phenylalanine means an ability to accumulate a significant amount of L-phenylalanine in a medium when the bacterium is cultured in the medium. Usually, it means an ability to accumulate not less than 0.5 g/l of L-phenylalanine under the conditions described in Examples mentioned below.
Examples of the phenylalanine analog are DL-p-fluorophenylalanine, m-fluorophenylalanine, xcex2-amino-xcex2-phenylpropionic acid, o-fluorophenylalanine, xcex2-2-thienylalanine, xcex2-3-thienylalanine, xcex2-2-furylalanine, xcex2-3-furylalanine, o-aminophenylalanine, p-aminophenylalanine, m-aminophenylalanine, xcex1-amino-xcex2-phenylethanesulfonate, xcex2-2-pyrrolalanine, 1-cyclopentene-1-alanine, 1-cyclohexene-1-alanine, xcex2-4-pyridylalanine, xcex2-4-pyrazolylalanine, p-nitro-phenylalanine, cyclohexylalanine, o-chlorophenylalanine, m-chlorophenylalanine, p-chlorophenylalanine, o-bromophenylalanine, m-bromophenylalanine, p-bromophenylalanine, xcex2-4-thiazolealanine and the like.
To be resistant to an phenylalanine analog means that the bacterium can grow in the presence of the phenylalanine analog in the amount at which the wild type strain (for example, strain AS-1) can not grow. The amount varies depending on the kinds of the phenylalanine analog. In the case of DL-p-fluorophenylalanine, it is usually 2 g/l under the conditions described in Examples mentioned below, and in the case of m-fluorophenylalanine, it is usually 1 g/l under the conditions described in Examples mentioned below.
It is preferred that the bacterium of the present invention is resistant to at least two kinds of the phenylalanine analog. For example, it is preferred that it is resistant to DL-p-fluorophenylalanine and m-fluorophenylalanine.
To be resistant to L-phenylalanine means that the bacterium can grow in the presence of L-phenylalanine in the amount at which the wild type strain (for example, strain AS-1) can not grow. The amount is usually 8 g/l under the conditions described in Examples mentioned below.
The bacterium of the present invention is preferably resistant to a higher concentration (for example, 0.1 M) of L-phenylalanine.
The bacterium of the present invention can be obtained by conferring the required resistances to a bacterium belonging to the genus Methylophilus in order. The order of conferring the phenylalanine analog resistance and the L-phenylalanine resistance is not limited and the conferring may be made in any order.
The method of obtaining a bacterium belonging to the genus Methylophilus which is resistant to the phenylalanine analog and a bacterium belonging to the genus Methylophilus which is resistant to L-phenylalanine will be explained below.
The bacteria belonging to the genus Methylophilus which is resistant to a phenylalanine analog can be obtained by culturing bacteria belonging to the genus Methylophilus in a minimal medium containing the phenylalanine analog at the growth inhibitory concentration and selecting a growing strain.
The selection of the phenylalanine-analog-resistant strain may be performed with one kind of the phenylalanine analog, or with more kinds of the phenylalanine analogs. The selection of the strain may be performed once or more for one kind of the phenylalanine analog.
An amount of the phenylalanine analog which is to be added to the medium depends on a kind of the phenylalanine analog, but is preferably not less than 2 g/L in the case of DL-p-fluorophenylalanine. The bacteria belonging to the genus Methylophilus may be subjected to a mutation treatment prior to the selection. Mutation may be performed by ultraviolet irradiation or by treatment with a mutagen usually used for artificial mutagenesis such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) or nitrous acid and the like.
The bacteria belonging to the genus Methylophilus which is resistant to L-phenylalanine can be obtained by culturing bacteria belonging to the genus Methylophilus in a minimal medium containing L-phenylalanine at the concentration that causes growth inhibition, and selecting a growing strain. Growth inhibition herein refers to slow growth or stop of growth. The selection of the strains may be performed once or more. A concentration of L-phenylalanine in the medium is not particularly limited, but it is exemplified by not less than 0.05 M, preferably 0.1 M. The bacteria belonging to the genus Methylophilus may be subjected to a mutation treatment prior to the selection in the same manner as described above.
The bacterium belonging to the genus Methylophilus which is resistant to L-phenylalanine, obtained as mentioned above, can grow in the presence of L-phenylalanine at a concentration at which its parent strain can not grow.
The bacterium of the present invention is preferably resistant to cinnamic acid.
To be resistant to cinnamic acid means that the bacterium can grow in the presence of cinnamic acid in the amount at which the wild type strain (for example, strain AS-1) can not grow. The amount is usually 50 mg/l under the conditions described in Examples mentioned below.
The bacteria belonging to the genus Methylophilus which is resistant to cinnamic acid can be obtained by a similar way to that for phenylalanine analog-resistant bacteria. The order of conferring the cinnamic acid resistance, the phenylalanine analog resistance and the L-phenylalanine resistance is not limited and the conferring may be made in any order.
L-Phenylalanine is concerned with several regulation steps on L-phenylalanine biosynthesis. Therefore, single mutation which causes L-phenylalanine resistance may be effective for improvement of L-phenylalanine productivity, however, it is preferably that more regulations are desensitized by double or more mutations. A bacterium belonging to the genus Methylophilus which has single mutation can be used as a starting source for breeding of an L-phenylalanine-producing strain, even though its productivity of L-phenylalanine is low.
The bacterium of the present invention may be enhanced in activity of one or more enzymes of L-phenylalanine biosynthetic pathway by usual mutation treatment or genetic engineering techniques.
For example, L-phenylalanine biosynthesis can be enhanced by increasing an ability to produce phosphoenol pyruvate in a bacterium belonging to the genus Methylophilus (International Publication No. WO 97/08333).
L-Phenylalanine productivity is improved by enhancing a desensitized chorismate mutase-prephenate dehydratase gene (Japanese Patent Application Laid-Open Nos. 5-236947 and 62-130693), and/or a desensitized 3-deoxyl-D-arabino-hepturosonic acid 7-phosphate synthase gene (Japanese Patent Application Laid-Open Nos. 5-236947 and 61-124375).
 less than 2 greater than  The Method of the Present Invention
The method of the present invention is a method for producing L-phenylalanine comprising cultivating the bacterium of the present invention in a culture medium, to produce and accumulate L-phenylalanine in a culture, and recovering L-phenylalanine from the culture.
The cultivation of the bacterium of the present invention may be performed by the method which is usually used for cultivation of methanol-assimilating microorganisms. The culture medium used in the present invention may be either a synthetic medium or a natural medium, so long as the medium includes a carbon source, a nitrogen source and mineral nutrients and, if necessary, other organic micronutrients.
If methanol is used as a main carbon source, L-phenylalanine can be produced inexpensively. Methanol is usually added to the culture medium usually in amount of 0.001 to 30% by weight, when it is used as a main carbon source. As the nitrogen source, ammonium sulfate may be used by adding it to the culture medium. In addition to these, a small amount of mineral nutrients such as potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate, and manganese sulfate may be used.
The cultivation is performed usually under aerobic conditions such as shaking culture and aeration and stirring culture, at pH of 5 to 9, and at a temperature of 20 to 40xc2x0 C. It is usually completed for 24 to 120 hours.
The culture includes cells and the culture medium, and is preferably the culture medium.
The recover of L-phenylalanine from the culture may be performed according to combination of known methods such as ionexchange resin method and precipitation method.